Calabrese, AN, Jackson, SM, Jones, LN et al. (10 more authors) (2017) Topological dissection of the membrane transport protein Mhp1 derived from cysteine accessibility and mass spectrometry. Analytical Chemistry, 89 (17). pp. 8844-8852. ISSN 0003-2700
Abstract
Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallised in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-L-hydantoin (L-BH) does not shift this conformational equilibrium, but, systematic co-addition of the two results in an attenuation of labelling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion-substrate coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilisation of structure. The methodology combines covalent labeling, mass spectrometry and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2017 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
Dates: |
|
Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Biomolecular Mass Spectroscopy (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds) |
Funding Information: | Funder Grant number BBSRC BB/E012558/1 BBSRC BB/K000659/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 24 Jul 2017 11:44 |
Last Modified: | 23 Jun 2023 22:33 |
Status: | Published |
Publisher: | American Chemical Society |
Identification Number: | 10.1021/acs.analchem.7b01310 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:119336 |